In this specification the parenthetic number indicates the reference listed below.
Recent whole genome sequence analyses revealed that a high degree of proteomic complexity is achieved with a limited number of genes. This surprising finding underscores the importance of alternative splicing, through which a single gene can generate multiple structurally and functionally distinct protein isoforms (1). Based on genome-wide analysis, 35-60% of human genes are thought to encode at least two alternatively spliced isoforms (2). The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing essential roles in many biological processes, such as embryonic development, cell growth, and apoptosis. Splicing mutations located in either intronic or exonic regions frequently cause hereditary diseases (reviewed in Refs. 3-5). More than 15% of mutations that cause genetic disease affect pre-mRNA splicing (6). Pre-mRNA splicing is also regulated in a tissue-specific or developmental stage specific manner. Indeed, the selection of splice site can be altered by numerous extracellular stimuli, including growth factors, cytokines, hormones, depolarization, osmotic shock, and UVC irradiation through synthesis, phosphorylation, and a change in localization of serine/arginine-rich (SR)1 proteins (7).
SR proteins are a family of essential factors required for constitutive splicing of pre-mRNA (8) and play an important role in modulating alternative splicing (9). They are highly conserved in eukaryotes and are characterized by having one or two RNA-recognition motifs at the amino terminus and an RS domain at the carboxyl terminus (10, 11). RS domains consist of multiple consecutive RS/SR dipeptide repeats and differ in length among different SR proteins. Extensive phosphorylation of serines in the RS domain occurs in all SR proteins (12, 13). Although its precise physiological role is still unknown, phosphorylation of SR proteins affects their protein-protein and protein-RNA interactions (14), intracellular localization and trafficking (15, 16), and alternative splicing of pre-mRNA (17). Spliceosome assembly may be promoted by phosphorylation of SR proteins that facilitate specific protein interactions, while preventing SR proteins from binding randomly to RNA (14). Once a functional spliceosome has formed, dephosphorylation of SR proteins appears to be necessary to allow the transesterification reactions to occur (18). Therefore, the sequential phosphorylation and dephosphorylation of SR proteins may mark the transition between stages in each round of the splicing reaction. To date, several kinases have been reported to phosphorylate SR proteins, including SRPK family kinases (19, 20), hPRP4 (21), and topoisomerase I (22), and a family of kinases termed Clk (Cdc2-like kinase), or LAMMER kinases from the consensus motif, consisting of four members (Clk 1/Sty and Clk2.4) (23, 24).
Mammalian Clk family kinases contain an SR domain and are demonstrated to phosphorylate SR proteins in vitro and SF2/ASF in vivo (24). Clks are shown to be dual-specificity kinases that autophosphorylate on tyrosine, serine, and threonine residues in overexpression systems and in vitro (24-26). When overexpressed, the catalytically inactive mutant kinases localize to nuclear speckles where splicing factors are concentrated, whereas the wild-type enzymes distribute throughout the nucleus and cause speckles to dissolve (23). The overexpression of Clks also affects splicing site selection of pre-mRNA of both its own transcript and adenovirus E1A transcripts in vivo (17). These results have led us to the current model that Clk family members regulate alternative splicing by phosphorylation of SR proteins, although their signal pathways and biological functions are largely unknown in vertebrates.